Type 2 diabetes mellitus (T2DM) is a major cause of morbidity and mortality among the U.S. population. Therefore, new and better ways to prevent and treat T2DM are urgently needed. Insulin resistance is one of the earliest and most significant abnormalities in the pathogenesis of T2DM. However, the molecular basis for the insulin resistance of T2DM is not fully understood. Toll-like receptor (TLR) 4 is a cell surface receptor that generates immune responses by activating a cascade of pro-inflammatory events. TLR4 is expressed on insulin-target (myocytes, hepatocytes, adipocytes) and inflammatory (monocytes/macrophages) cells. This receptor is activated by bacteria-derived lipopolysaccharide (LPS) and by saturated free fatty acids (FFA). Studies from various groups, including ours, have shown that TLR4 expression is elevated in muscle and inflammatory cells from insulin resistant subjects. Several studies also have demonstrated that the plasma concentration of two TLR4 ligands, FFA and LPS, is increased in obese and T2DM subjects. Moreover, emerging evidence from cell culture and animal studies suggest that TLR4 and downstream pathways [MAPKs and nuclear factor (NF)?B] could play an important role in the pathogenesis of insulin resistance and T2DM. Despite the considerable body of data linking TLR4 with insulin resistance, it is not known whether TLR4 is directly involved in the pathogenesis of insulin resistance in obese and T2DM human subjects. In this study, we will utilize a specific TLR4 inhibitor in vivo with the goals to (i) determine the role that TLR4 signalng plays in the pathogenesis of insulin resistance and T2DM in humans; and (ii) elucidate the mechanism/s by which TLR4 mediates insulin resistance in human subjects. We propose two Aims to test the following hypotheses: (1) Pharmacologic inhibition of TLR4 with E5564 will prevent lipid-induced insulin resistance in lean normal glucose-tolerant subjects; and (2) Pharmacologic inhibition of TLR4 with E5564 will reduce inflammation and improve glucose metabolism in insulin resistant (obese nondiabetic and obese T2DM) subjects. The results from this study will advance our understanding about the pathophysiology of insulin resistance in human subjects, and, if positive, will validate TLR4 as a viable target for T2DM prevention and treatment.